CN104937015A - Method for recycling plastic products - Google Patents

Abstract

The invention relates to a method for recycling at least one plastic product, the method comprising degrading at least one polymer of the plastic product to monomers using an enzyme and recovering the resulting monomers. The method of the invention may be used for degrading, simultaneously or sequentially at least two different polymers of the plastic product, and/or for recycling at least two plastic products.

Description

Method for recycling plastic products

technical field

The present invention relates to a method for recycling plastic articles such as waste plastic. More particularly, the present invention relates to a biological process for depolymerizing at least one polymer of a plastic article and recovering the resulting monomers, which can be further reprocessed to synthesize new polymers and manufacture new plastic articles.

Background technique

Plastic is an inexpensive and durable material that can be used to manufacture a wide variety of products for a wide range of applications, resulting in a dramatic increase in the manufacture of plastics over the past decade. About 40% of these plastics are used in single-use applications such as packaging, agricultural films, single-use consumer products or for short-term products that are discarded within a year of manufacture. Because of the durability of the polymers involved, large quantities of plastics accumulate in landfills and natural habitats around the world, creating increasing environmental concerns. Even degradable and biodegradable plastics can persist for decades depending on local environmental factors such as UV exposure levels, temperature, presence of suitable microorganisms, etc.

One approach to reducing the environmental and economic impact associated with the accumulation of plastics is closed-loop recycling in which plastic materials are mechanically reprocessed to make new products. For example one of the most common closed loop recycling is the recycling of polyethylene terephthalate (PET). Continuous processing of PET waste to produce food contact approved recycled PET (rPET) which is collected, sorted, pressed into bales, crushed, washed, flaked, melted and extruded in pellet form out and sold. This recycled PET can then be used to make fabrics for the clothing industry or new packaging such as bottles or blister packs.

However, plastic waste is often collected together so that the plastic bale contains a mixture of different plastics, the composition of which may vary from source to source, and the proportion of which may vary from bale to bale. Therefore, recycling methods require an initial selection to classify plastic articles according to their composition, size, resin type, color, functional additives used, etc.

In addition, the actual plastic recycling method uses a lot of electricity, especially during the extrusion step, and the equipment used is also expensive, resulting in a potentially uncompetitively high price compared to virgin plastic.

Another possible method for recycling plastics consists of chemical recycling that allows recovery of the chemical components of the polymers. The resulting monomers can then be used to remake plastics or make other synthetic chemicals. However, to date, this recycling method has only been performed on purified polymers and is not effective on raw plastics consisting of mixtures of crystalline and amorphous polymers with additives.

Therefore, there is a need for an upcycling method for recycling plastic articles, which does not require initial sorting and costly pretreatment, and which can be used to recycle different plastic materials.

Contents of the invention

The inventors now propose a biological method for depolymerizing at least one polymer of at least one plastic article with low energy consumption. The method of the present invention allows the recovery of the monomers forming the original polymers of the plastic articles so that they can be reprocessed to synthesize new polymer chains of the same type. More particularly, the inventors propose to use specific enzymes capable of depolymerizing the polymers of the plastic article and producing a mixture of monomers forming the original polymer.

In this regard, it is an object of the present invention to propose a method for recycling at least one plastic article, said method comprising using an enzyme to depolymerize at least one polymer of the plastic article into monomers and recovering the resulting monomers .

Another object of the invention relates to a method for recycling at least two different polymers of plastic articles, wherein said at least two different polymers are depolymerized simultaneously or sequentially, and wherein the resulting monomers are recovered.

The invention also relates to a method for recycling at least two different plastic articles simultaneously or sequentially, wherein at least one polymer of each plastic article is degraded into monomers using at least one enzyme, and wherein the resulting monomers are to recycle.

Preferably, said plastic article comprises at least one polymer selected from polyesters and polyamides.

More preferably, the polyester is selected from the group consisting of polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyterephthalate Ethylene glycol isosorbide phthalate (PEIT), polylactic acid (PLA), poly(L-lactic acid) (PLLA), poly(D-lactic acid) (PDLA), poly(D,L-lactic acid) (PDLLA ), PLA stereocomplex (scPLA), polyhydroxyalkanoate (PHA), poly(3-hydroxybutyrate) (P(3HB)/PHB), poly(3-hydroxyvalerate) (P( 3HV)/PHV), poly(3-hydroxyhexanoate) (P(3HHx)), poly(3-hydroxyoctanoate) (P(3HO)), poly(3-hydroxydecanoate) (P( 3HD)), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)/PHBV), poly(3-hydroxybutyrate-co-3-hydroxyhexyl ester) (P(3HB-co-3HHx)/(PHBHHx)), poly(3-hydroxybutyrate-co-5-hydroxyvalerate) (PHB5HV), poly(3-hydroxybutyrate-co- -3-hydroxypropionate) (PHB3HP), polyhydroxybutyrate-co-hydroxyoctanoate (PHBO), polyhydroxybutyrate-co-hydroxyoctadecanoate (PHBOd), poly(3-hydroxy Butyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) (P(3HB-co-3HV-co-4HB)), polybutylene succinate (PBS), poly Butylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furoate (PEF), polycaprolactone (PCL), poly (ethylene adipate) (PEA), and blends/mixtures of these materials.

And said polyamide is preferably selected from: polyamide-6 or poly(β-caprolactam) or polycaprolactam (PA6), polyamide-6,6 or poly(hexamethylene adipamide) (PA6,6), Poly(11-aminoundecylamide) (PA11), polydodecanolactam (PA12), poly(butylene adipamide) (PA4,6), poly(pentamethylene sebacylamide) (PA5 ,10), poly(hexamethylene azelamide) (PA6,9), poly(hexamethylene sebacamide) (PA6,10), poly(hexamethylene dodecamide) (PA6,12), Poly(m-xylylene adipamide) (PAMXD6), polyhexamethylene adipamide/polyhexamethylene terephthalamide copolymer (PA66/6T), polyhexamethylene adipamide/polymethylene Hexamethylene phthalamide copolymer (PA66/6I), and blends/mixtures of these materials.

Advantageously, the recovered monomers are further reprocessed to synthesize new polymers.

Preferably, said enzyme is a degradative enzyme suitable for depolymerizing at least one polymer of said plastic article into monomers.

The degrading enzyme is preferably selected from: cutinase (EC 3.1.1.74), lipase (EC 3.1.1.3), esterase, carboxylesterase (EC 3.1.1.1), p-nitrobenzyl esterase, serine Protease (EC 3.4.21.64), protease, amidase, aryl acyl amidase (EC 3.5.1.13), oligomer hydrolase such as 6-aminocaproate cyclic dimer hydrolase (EC 3.5.2.12 ), 6-aminocaproate dimer hydrolase (EC 3.5.1.46), 6-aminocaproate-oligomer hydrolase (EC 3.5.1.B17), peroxidase, laccase (EC 1.10.3.2).

In another particular embodiment, said enzyme may be an intermediate enzyme producing and/or activating at least one intermediate molecule suitable for depolymerizing at least one polymer of a plastic article into monomers.

In a specific embodiment, the method comprises the steps of:

a) contacting said plastic article with at least one microorganism expressing and secreting a depolymerase or an intermediate enzyme;

b) recycling monomers resulting from the depolymerization of at least one polymer of the plastic article.

In a particular embodiment, said microorganism expressing and secreting said enzyme is a recombinant microorganism with an improved metabolism preventing consumption of the resulting monomer.

In another specific embodiment, the microorganism is a recombinant microorganism expressing and secreting a recombinant degrading enzyme.

In another specific embodiment, the method comprises the steps of:

a) contacting said plastic article with at least one depolymerase;

b) recycling monomers resulting from the depolymerization of at least one polymer of the plastic article.

According to the invention, the degradative enzyme can be used together with at least one lipophilic and/or hydrophilic agent.

Plastic articles can be pretreated prior to degradation. More particularly, pretreatment may include mechanical/physical modification of plastic articles such as cutting and impacting, crushing and grinding, fractionation, cryogenic cooling steps, drying, dehydration, agglomeration or granulation.

In a particular embodiment, the plastic article may be further sorted, washed and/or biologically cleaned prior to degradation.

These and other objects and embodiments of the invention will become more apparent after a detailed description of the invention including preferred embodiments of the invention given in general terms.

Description of drawings

Figure 1 shows the production of lactic acid after hydrolysis of a polylactic acid polymer contained in plastic pellets according to the method of the invention. Adjusting the pH to keep it around 8 allows increased monomer production even after 48 or 72 hours;

Figure 2 shows that polyethylene terephthalate contained in plastic articles can be hydrolyzed and terephthalic acid monomer can be recovered by the method of the present invention;

Figure 3 shows the effect of the particle size of the PET plastic product on the efficiency of the process of the invention.

detailed description

The invention relates to a complete recycling method for recycling plastic articles by depolymerizing at least one polymer constituting said plastic articles, wherein a repolymerizable monomer mixture is produced and recovered.

definition

This disclosure will be best understood by reference to the following definitions.

In the context of the present invention, the term "plastic article" means an article made of at least one plastic material comprising at least one polymer, such as plastic sheet, plastic tube, plastic rod, plastic profile, plastic profile, plastic block, plastic Any article made of fibers etc. and possibly other substances or additives such as plasticizers, mineral or organic fillers. Preferably, the plastic article consists of semicrystalline and/or amorphous polymers, or mixtures of semicrystalline polymers and additives. More preferably, the plastic article is a manufactured article such as packaging, agricultural film, disposable items, and the like. The plastic materials of the present invention include synthetic, degradable and biodegradable plastics. In the context of the present invention, natural rubber and synthetic rubber are not considered plastic materials and rubber products are excluded from the scope of the present invention.

"Polymer" refers to a chemical compound or mixture of compounds whose structure consists of a plurality of repeating units linked by covalent chemical bonds. In the context of the present invention, the term polymer includes natural or synthetic polymers which consist of repeating units of one type (i.e. homopolymers) or of a mixture of different repeating units (i.e. block copolymers and random copolymers). ).

A "recycling process" with respect to a plastic article refers to a process by which at least one polymer of said plastic article is degraded to produce repolymerizable monomers which are recovered for reuse.

In this specification, "recombinant microorganism" refers to a microorganism whose genome has been improved by the insertion of at least one nucleic acid sequence or unit. Typically, the inserted nucleic acid sequence or unit is not naturally present in the genome of the microorganism. The nucleic acid sequences or units are assembled and/or inserted into the microorganism or its progenitors using recombinant DNA technology (also known as gene cloning or molecular cloning), which refers to the extraction of DNA from an organism The technique of transferring to another organism. The nucleic acid sequence or unit may be integrated into the microbial chromosome, or may be present on a plasmid. A "recombinant microorganism" also refers to a microorganism whose genome has been modified by inactivation or deletion of at least one nucleic acid sequence or unit. The resulting recombinant microorganisms can be produced by various methods and, once produced, can replicate without the use of additional recombinant DNA techniques. Otherwise, recombinant microorganisms can be generated from metagenomic libraries.

The terms "nucleic acid", "nucleic acid sequence", "polynucleotide", "oligonucleotide" and "nucleotide sequence" are used interchangeably and refer to a sequence of deoxynucleotides and/or nucleotides. Nucleotide sequences can first be prepared, for example, by recombinant, enzymatic and/or chemical techniques, and then replicated in host cells or in vitro systems. The nucleotide sequence preferably comprises an open reading frame encoding a (poly)peptide. The nucleotide sequence may contain additional sequences such as transcription terminators, signal peptides, introns, and the like.

In the context of the present invention, the term "derived from a microorganism" with respect to an enzyme or (poly)peptide indicates that the enzyme or (poly)peptide is isolated from such a microorganism, or that the enzyme or (poly)peptide comprises All or a biologically active part of the amino acid sequence of a characterized enzyme or (poly)peptide.

The term "vector" refers to a DNA molecule used as a vehicle to transfer recombinant genetic material into a host cell. The main types of vectors are plasmids, bacteriophages, viruses, cosmids and artificial chromosomes. The vector itself is usually a DNA sequence consisting of an insert (heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector to transfer genetic information to a host is usually to isolate, multiply or express the insert in target cells. Vectors, known as expression vectors (expression constructs), are specifically adapted for the expression of heterologous sequences in target cells, and generally have a promoter sequence driving expression of the heterologous sequence encoding a polypeptide. Typically, regulatory elements present in expression vectors include transcriptional promoters, ribosomal binding sites, terminators and, optionally, operators. Preferably, the expression vector also includes an origin of replication for autonomous replication in the host cell, a selectable marker, a limited number of useful restriction sites and the possibility of high copy number. Examples of expression vectors are cloning vectors, modified cloning vectors, specially designed plasmids and viruses. Expression vectors that provide suitable levels of expression of polypeptides in various hosts are well known in the art. Bacterial expression vectors well known in the art include pET11a (Novagen), lamda gt11 (Invitrogen).

Expression vectors can be introduced into host cells using standard techniques. Examples of such techniques include transformation, transfection, lipotransfection, protoplast fusion, and electroporation. Examples of techniques for introducing nucleic acids into cells and expressing nucleic acids to make proteins are provided in, for example, Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998 and Sambrook et al., Molecular cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.

plastic products

The present invention proposes to degrade plastic articles down to the monomer level so that the monomers can be reused to repolymerize polymers and further manufacture new plastic articles.

The method of the invention can be used to recycle plastic articles made from several different plastic materials. For example, a plastic article may comprise successive layers of different plastic materials.

The recycling method of the present invention can be used to degrade all kinds of plastic articles without the need for preliminary plastic sorting and/or cleaning. More particularly, the method of the invention can be directly applied to plastic articles from plastic waste collection. For example, the method of the present invention can be applied to a mixture of household plastic waste including plastic bottles, plastic bags, plastic packaging, textile waste and the like.

The plastic articles that are the object of the method of the present invention may comprise different kinds of plastic materials, including synthetic plastic materials derived from petrochemical products, or bio-based plastic materials (ie entirely or substantially composed of biological products).

The target plastic product can contain one or several polymers, and additives. A plastic article can consist of several polymers arranged in different layers or fused together. Furthermore, plastic articles can be composed of semi-crystalline polymers or mixtures of semi-crystalline and amorphous polymers as well as additives.

In a particular embodiment, the plastic article consists only of polymers comprising a saturated linear carbon backbone, which polymers may also comprise saturated or unsaturated rings, such as aromatic rings.

In a particular embodiment, the target plastic article comprises polyester and/or polyamide. Preferably, the plastic article contains only polyester and/or polyamide. Preferred polyesters are polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene terephthalate Isosorbide (PEIT), polylactic acid (PLA), poly(L-lactic acid) (PLLA), poly(D-lactic acid) (PDLA), poly(D,L-lactic acid) (PDLLA), PLA stereocomplex (scPLA), polyhydroxyalkanoate (PHA), poly(3-hydroxybutyrate) (P(3HB)/PHB), poly(3-hydroxyvalerate) (P(3HV)/PHV), Poly(3-hydroxyhexanoate) (P(3HHx)), poly(3-hydroxyoctanoate) (P(3HO)), poly(3-hydroxydecanoate) (P(3HD)), poly( 3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)/PHBV), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P( 3HB-co-3HHx)/(PHBHHx)), poly(3-hydroxybutyrate-co-5-hydroxyvalerate) (PHB5HV), poly(3-hydroxybutyrate-co-3-hydroxypropionate ester) (PHB3HP), polyhydroxybutyrate-co-hydroxyoctanoate (PHBO), polyhydroxybutyrate-co-hydroxyoctadecanoate (PHBOd), poly(3-hydroxybutyrate-co- 3-Hydroxyvalerate-co-4-hydroxybutyrate) (P(3HB-co-3HV-co-4HB)), polybutylene succinate (PBS), polysuccinate adipate Butylene glycol ester (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furoate (PEF), polycaprolactone (PCL), poly(ethylene adipate alcohol ester) (PEA), and blends/mixtures of these materials; and the preferred polyamide is polyamide-6 or poly(ε-caprolactam) or polycaprolactam (PA6), polyamide-6,6 or poly (Hexamethylene adipamide) (PA6,6), poly(11-aminoundecylamide) (PA11), polydodecanolactam (PA12), poly(butylene adipamide) (PA4, 6), poly(pentamethylene sebacamide) (PA5,10), poly(hexamethylene azelate) (PA6,9), poly(hexamethylene sebacamide) (PA6,10), poly( Hexamethylene dodecamide) (PA6,12), poly(m-xylylene adipamide) (PAMXD6), polyhexamethylene adipamide/polyhexamethylene terephthalamide copolymer (PA66 /6T), polyhexamethylene adipamide/polyhexamethylene isophthalamide copolymer (PA66/6I), and blends/mixtures of these materials.

In a particular embodiment, the plastic article is composed of an aliphatic polyester such as polylactic acid, and more particularly semi-crystalline polylactic acid.

In another embodiment, the plastic article is made of aromatic polyesters such as polyethylene terephthalate and/or polytrimethylene terephthalate, more particularly semi-crystalline polyethylene terephthalate and/or polytrimethylene terephthalate.

plastic degradation

It is an object of the present invention to provide degrading enzymes suitable for hydrolyzing chemical bonds between monomers of at least one polymer of plastic articles.

According to the invention, depending on the polymer to be hydrolyzed, such degrading enzymes may be cutinases, lipases, esterases, carboxylesterases, p-nitrobenzyl esterases, serine proteases, proteases, amidases, aryl Acylamidase, oligomer hydrolase, peroxidase, laccase, etc.

For example, serine proteases (such as proteinase K from Tritirachium album or PLA depolymerase from Amycolatopsis sp.) or lipases (such as from Candida antarctica (Candida antarctica) or cryptococcus (Cryptococcus sp.) or Aspergillus niger (Aspergillus niger) lipases) or esterases (such as those from Thermobifida halotolerans) are used to depolymerize plastics containing polylactic acid (PLA). Cutinase (such as from Thermobifida fusca or Thermobifida alba or Fusarium solani pisi) or lipase (such as source Lipase PS from Burkholderia cepacia was used to depolymerize plastics comprising PET or PTT. Cutinases (such as those derived from Fusarium solani) or aryl acylamidases (such as those derived from Nocardia farcinica) or oligomers can be Hydrolase (such as 6-aminocaproate oligomer hydrolase from Arthrobacter sp.) or amidase (such as amidase from Beauveria brongniartii) for depolymerization Plastic products containing PA6 or PA6,6.

In a particular embodiment, the plastic articles to be recycled are contacted with degradative enzymes, which may be natural or synthetic.

For example, degradative enzymes can be produced by recombinant techniques, or when naturally occurring, they can be isolated or purified from natural sources, or they can be produced artificially. Enzymes can be in soluble form, or in a solid phase. In particular, they can be bound to cell membranes or lipid vesicles, eg in the form of beads, columns, plates, etc., or to synthetic supports such as glass, plastic, polymers, filters, membranes.

The enzyme is preferably in isolated or purified form. Preferably, the enzymes of the invention are expressed, derived, secreted, isolated or purified from microorganisms. Enzymes can be purified by techniques known per se in the art and stored under conventional techniques. Enzymes can be further modified to increase, for example, their stability or activity.

In another embodiment, plastic articles to be recycled are contacted with microorganisms that synthesize and secrete degradative enzymes. In the context of the present invention, the enzyme may be secreted into the culture medium or into the cell membrane of the microorganism where the enzyme may be anchored.

The microorganism may naturally synthesize the degradative enzyme, or it may be a recombinant microorganism into which a recombinant nucleotide sequence encoding the degradative enzyme is inserted using, for example, a vector.

For example, a nucleotide molecule encoding a target degrading enzyme is inserted into a vector such as a plasmid, a recombinant virus, a phage, an episome, an artificial chromosome, and the like. Advantageously, the nucleotide molecule is under the control of a specific promoter. The vector is then transfected into a host microorganism to form a recombinant microorganism. The host is further cultured under culture conditions suitable for the host, thereby obtaining a recombinant cell comprising the enzyme of the present invention. Culture conditions suitable for the host are well known to those skilled in the art.

The nucleotide molecules of the invention may be in isolated or purified form and prepared, isolated and/or manipulated by techniques known per se in the art, such as cloning and expression of cDNA libraries, amplification, enzymatic synthesis or recombinant techniques. Nucleotide molecules can also be synthesized in vitro by well known chemical synthesis techniques. The nucleotide molecules of the invention may comprise additional nucleotide sequences such as regulatory regions, i.e. promoters, enhancers, silencers, terminators which can be used to cause or regulate the expression of the enzyme in the host cell or system of choice. wait.

Recombinant microorganisms can be used directly. Alternatively, or in addition, the recombinant enzyme may be purified from the culture medium. Any usual separation/purification means such as salting out, gel filtration, hydrophobic interaction chromatography or ion exchange chromatography can be used for this purpose.

In certain embodiments, microorganisms known to synthesize and secrete degradative enzymes may be used. For example, Aspergillus oryzae, Humicola insolens, Penicillium citrinum, Fusarium solani and Thermobifida cellulolysitica, which synthesize and secrete cutinases, can be used to degrade PET-containing plastic articles. In the same way, Candida antarctica/Thermomyces lanuginosus, Burkholderia sp. and Triticum aestivum synthesize lipases that depolymerize PET. Amycolatopsis K104-1 and K104-2, Candida albicans ATCC 22563, Paenibacillus amylolyticus (Paenibacillus amylolyticus) TB-13, desert cysts (Kibdelosporangium aridum) JCM7912, Waiwai Enders Saccharothrix waywayandensis JCM 9114, Amycolatopsis orientalis IFO 12362, and Actinomadura keratinilytica T16-1 were used to degrade plastic products containing PLA. Aspergillus fumigatus NKCM1706, Bionectria ochroleuca BFM-X1 can be used to degrade plastic articles comprising PBS. Thermomonospora fusca K13g and K7a-3, Isaria fumosorosea NKCM1712 can be used to degrade plastics containing PBAT. Bjerkandera adusta, which produces manganese peroxidase, can be used to degrade PA-containing plastic articles.

According to the invention, several microorganisms and/or purifying enzymes and/or synthetizing enzymes can be used together or sequentially to depolymerize different kinds of polymers contained in the same plastic article or in different plastic articles.

Advantageously, the microorganisms of the invention display an improved metabolism in order to prevent the consumption of monomers obtained by degradation of polymers. For example, the microorganism is a recombinant microorganism in which an enzyme that degrades the monomer has been deleted or knocked out. In addition, the method of the invention may be performed in a medium comprising at least one carbon source available to the microorganism such that said microorganism preferentially consumes this carbon source rather than the monomer.

Advantageously, the plastic article is brought into contact with a medium comprising microorganisms, glucose etc. as a carbon source, and a nitrogen source assimilable by the microorganisms, including organic nitrogen sources (e.g. peptone, meat extract, yeast extract, corn steep liquor) or inorganic nitrogen sources (eg, ammonium sulfate, ammonium chloride). If necessary, the medium may further contain inorganic salts (for example, sodium ions, potassium ions, calcium ions, magnesium ions, sulfate ions, chloride ions, phosphate ions). In addition, the medium can be supplemented with trace components such as vitamins, oligo-elements and amino acids.

Recycling Method Parameters

According to the invention, plastic articles can be recycled by contacting said plastic articles with a degradative enzyme targeting at least one polymer of said plastic article and/or with microorganisms that synthesize and secrete such degradative enzymes.

The method of the invention is particularly useful for degrading semi-crystalline polymers contained in plastic articles comprising said semi-crystalline polymer and ultimately one or several other semi-crystalline and/or amorphous polymers and/or or additives.

In a specific embodiment, the plastic article may be initially treated to physically alter its structure, thereby increasing the contact surface between the polymer and the enzyme. For example, a plastic article can be converted into an emulsion or powder which is added to a liquid medium containing microorganisms and/or enzymes. Additionally, plastic articles may be mechanically ground, pelletized, pelletized, etc. to reduce the shape and size of the material prior to addition to the liquid medium containing the microorganisms and/or enzymes.

The time required for the degradation of a plastic article can vary with the plastic article itself (i.e., the nature and origin of the plastic article, its composition, shape, etc.), the type and amount of microorganisms/enzymes used, and various process parameters (i.e., temperature, pH, additives, etc.) A person skilled in the art can easily adapt the method parameters to the plastic and/or the degrading enzyme.

Advantageously, the method is carried out at a temperature between 20°C and 80°C, more preferably at a temperature between 25°C and 60°C. Preferably, the temperature is maintained between 25°C and 50°C at least during the depolymerization step. More generally, the temperature is maintained below the inactivation temperature, which corresponds to the temperature at which the enzyme is inactive and/or the microorganism no longer synthesizes the degrading enzyme. Surprisingly, the inventors have found that the process of the invention can be carried out at temperatures below the Tg of the target polymer. According to the invention, the enzyme used in the depolymerization step may be added in an amount of at least 0.005% by weight, preferably at least 0.1% by weight and more preferably at least 1% by weight of the plastic article. And the added amount is advantageously at most 15% by weight and more preferably at most 5% by weight of the plastic article. Advantageously, the amount of degrading enzymes is in the range of 0.005% to 15% by weight of the plastic article, preferably in the range of 0.1% to 10% by weight, and more preferably in the range of 1% to 5% by weight.

The pH of the medium can be in the range of 4-10. Advantageously, the pH is adjusted according to the polymer/enzyme pair of interest to increase process efficiency. More particularly, the pH is adjusted to remain at the optimum pH for the enzyme. Indeed, depolymerization of polyesters and polyamides produces acidic monomers that cause a drop in pH. Addition of dilute base can be used to compensate for this acidification and maintain the pH at an optimal pH.

In a specific embodiment, at least lipophilic and/or hydrophilic agents are added to the medium to improve the depolymerization step. Inducers such as gelatin can be added to the medium to increase enzyme production. Surfactants such as Tween can be added to the medium to adjust the interfacial energy between the polymer and enzyme or microorganism and improve the degradation efficiency. Organic substances can be used to swell the polymer and increase its accessibility to microorganisms or enzymes.

Advantageously, the method of the invention is carried out without any degradation accelerators. In a particular embodiment, the method of the invention is carried out in a degradation liquid comprising only degradative enzymes and water. In a particular embodiment, the process of the invention is carried out in the absence of organic solvents.

The reaction time for depolymerizing the at least one polymer of the plastic article is advantageously between 5 and 72 hours. Such a reaction time allows the depolymerization to proceed sufficiently without being economically disadvantageous.

Treatment and reuse of recovered monomers

The mixture of monomers obtained from the depolymerization of the target polymer can be recovered sequentially or continuously at the end of the depolymerization step. Depending on the polymer being depolymerized and/or the recycled plastic article, a single monomer or several different monomers can be recovered.

The monomers can be further purified and conditioned in a repolymerizable form using all suitable purification methods. Examples of purification methods include stripping methods, separation by aqueous solution, selective condensation of steam, filtration after biological treatment and concentration of culture medium, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, Concentration and acid addition Dehydration and precipitation, nanofiltration, acid catalyst treatment, distillation in semi-continuous mode or continuous mode, solvent extraction, evaporative concentration, evaporative crystallization, liquid/liquid extraction, hydrogenation, azeotropic distillation methods, adsorption, column chromatography , simple vacuum distillation and microfiltration, these methods can be used in combination or alone.

The repolymerizable monomers can then be reused to synthesize polymers. Advantageously, polymers of the same nature are repolymerized. However, recycled monomers can be mixed with other monomers and/or oligomers to synthesize new copolymers.

In a specific embodiment, the repolymerization is carried out using a hydrolase under suitable conditions to allow polymerization. Initiators may be added to the monomer solution to aid polymerization. A person skilled in the art can readily adapt the process parameters to the monomers and polymers to be synthesized.

Other aspects and advantages of the invention will be disclosed in the following examples, which should be considered as illustrative and not as limiting the scope of the application.

Example

A] Aliphatic polyester recycling using enzymes

Thanks to the method of the invention, plastic articles comprising aliphatic polyesters such as PLA can be recycled. This experiment shows the recovery of lactic acid by treating plastic articles composed of semi-crystalline PLA with proteinase K.

Plastics and pretreatment

PLA pellets were purchased from NaturePlast (PLLA 001) and ground to a powder with a particle size of less than 500 μm using a universal mill ConduxCUM 100.

In an aluminum pan, at a scan rate of 10°C/min, under a nitrogen atmosphere (50mL/min), from -50°C to 300°C, using a Q 100TA-RCS 90 instrument, for a sample of about 8mg, using a differential scan Calorimetry (DSC) tests to determine the glass transition temperature (Tg) and crystallinity of polymers in plastic articles.

PLA powder has a Tg of 59°C and is semi-crystalline with a crystallinity of 14.9%.

Hydrolysis reaction

Hydrolysis of PLA powder was performed using proteinase K from Candida albicans (Sigma) to recover lactic acid. Enzyme solutions were prepared at a concentration of 10 mg/mL in Tris 20 mM pH 8 with CaCl ₂ 5 mM. During 7 days, 20 mg PLA was treated with 200 μg proteinase K in Tris HCl 20 mM pH 8 with 5 mM CaCl ₂ in a final volume of 5 mL at 37° C. using magnetic stirring.

In the experiments, the pH was maintained at pH 8 (corresponding to the optimal pH for proteinase K) with NaOH 0.5 M during the incubation to compensate for the acidification caused by lactic acid production. The experiment was similarly performed three times.

i) PLA in buffer without enzyme; ii) enzyme in buffer without PLA were used as controls.

Lactic Acid (LA) Analysis

Sample 160 μL of reaction medium at each analysis. The samples were centrifuged at 16000g for 3 minutes at 0°C. The supernatant for analysis was subjected to 0.45 μm filtration and 20 μL was injected into the HPLC. The HPLC used was Ultimate-3000 (Dionex, ThermoScientific) with an autosampler thermostat controlled to 10°C and a column chamber thermostat controlled to 50°C. For the analysis of LA, an Aminex H+HPX-87H column was used. Analysis was performed using ₅ mM _H2SO4 as eluent. The flow rate was set at 0.5 mL/min and the column was maintained at a temperature of 50°C. Detection of LA was performed at 220 nm using a variable wavelength detector. Quantitation was possible considering standards prepared with L-lactic acid (L-1750) from Sigma dissolved in Tris HCl 20 mM pH 8, in the concentration range 0-300 mM.

result

PLA powder was hydrolyzed by proteinase K and lactic acid was recovered. Lactate was not detected in the controls. As shown in Figure 1, the maximum concentration of lactic acid was obtained after 72 hours of hydrolysis when no pH adjustment was achieved. When the pH was controlled, the lactic acid concentration remained increased until 7 days of reaction. Maintaining the pH at pH 8 during hydrolysis allowed recovery of 5.87 ± 0.92 mM LA at 72 h, in contrast, 3.47 ± 0.57 mM LA was recovered without pH adjustment. Thus, the pH adjustment can thus be used as a parameter for adjusting the efficiency of the process.

B] Aromatic polyester recycling using enzymes

Thanks to the method according to the invention, plastic articles comprising aromatic polyesters such as PET and/or PTT can be recycled. This experiment shows the recovery of terephthalic acid by processing plastic articles comprising PET and/or PTT.

Plastics and pretreatment

Use different substrates:

-PET film, purchased from Goodfellow (ES 301445), thickness 0.25mm, is amorphous

-PTT pellets, purchased from DuPont (DuPont) ( 3301 NC010)

-PET bottle (previously contained in the trademark under the mineral water)

The PET film was cut into 10 mg (approximately 0.5 cm x 1 cm) pieces. It was washed in three consecutive steps to remove any protein or lipid contamination: in the first step, it was washed with 5g/L Triton-X 100; in the second step, it was washed with 100mM Na ₂ CO ₃ It is washed; finally, it is washed with deionized water. Each wash step was performed at 50°C for 30 minutes. Then, the PET film was dried with compressed air.

PTT was ground into a powder by using a cutting mill SM-2000 (Retsch) during 5 minutes, and then sieved with a sieve AS 200 (Retsch) with an amplitude of 1.5 mm during 10 minutes to obtain a powder of 1 mm.

The whole bottle was pretreated to increase the contact surface between PET and enzyme. During 5 minutes, they were mechanically ground into powders of different particle sizes by using a cutting mill SM-2000 (Retsch). Then, the collected powder was sieved with a sieve AS 200 (Retsch) at an amplitude of 1.5 mm during 10 minutes to obtain 3 powders with particle sizes of 1 mm, 500 μm and 250 μm, respectively.

In an aluminum pan, at a scan rate of 10°C/min, under a nitrogen atmosphere (50mL/min), from -50°C to 300°C, using a Q 100TA-RCS 90 instrument, for a sample of about 8mg, using a differential scan Calorimetry (DSC) tests to determine the glass transition temperature (Tg) and crystallinity of polymers in plastic articles.

The PTT and PET bottle powders have Tg's of 50.6°C and 77.2°C, respectively, and are semi-crystalline with crystallinity of 36% and 30%, respectively.

cutinase manufacturing

Thermobifida cellulosilytica DSM44535 was obtained from the German Resource Center for Biological Material (DSMZ, Germany). Strains were maintained on LB agar plates and cultured in 500 mL shake flasks (200 mL LB medium) at 37°C and 160 rpm for 24 hours. Cells were harvested by centrifugation at 3200g and 4°C for 20 minutes.

The vector pET26b(+) (Novagen, Germany) was used to express the cutinase THC_Cut2 from Thermobifida cellulosytica in Escherichia coli BL21-Gold (DE3) (Stratagene, Germany).

The cutinase-encoding gene Thc_cut2 was amplified from genomic DNA of T. cellulosilytica DSM44535 by standard polymerase chain reaction (PCR). Based on the known sequence of the gene encoding cutinase from T. fusca YX (Genbank accession numbers YP_288944 and YP_28894333), two primers 5'-CCCCCGCTCATATGGCCAACCCCTACGAGCG-3' (SEQ ID 1 - forward primer) and 5'- GTGTTCTAAGCTTCAGTGGTGGTGGTGGTGGTGCTCGAGTGCCAGGCACTGAGAGTAGT-3' (SEQ ID 2 - reverse primer), allowing amplification of the respective genes without signal peptide and introduction of 6xHis-Tag at the C-terminus of cutinase. Designed primers containing the gene for cloning into the vector pET26b The restriction sites NdeI and HindIII in . PCR was performed in a volume of 50 μL using genomic DNA as template, 0.4 μM of each primer, 0.2 mM dNTP, 5 units of Phusion DNA polymerase (Finnzymes) and 1X reaction buffer provided by the supplier. PCR was performed in a Gene Amp PCR 2200 thermal cycler (Applied Biosystems, USA). 35 cycles were performed, each cycle sequentially exposing the reaction mixture to 98°C (30s, denaturation), 63°C (30s, annealing) and 72°C (30s, extension). Plasmids and DNA fragments were purified by Qiagen DNA purification kit (Qiagen, Germany). Purified amplified PCR products thus obtained were digested with restriction endonucleases NdeI and HindIII (New England Biolabs, USA) and dephosphorylated with alkaline phosphatase (Roche, Germany) according to the manufacturer's instructions. Ligation to pET26b using T4 DNA-ligase (Fermentas, Germany) And transformed in Escherichia coli BL21-Gold (DE3).

The sequence of the gene was determined by DNA sequencing using primers 5'-GAGCGGATAACAATTCCCCTTAGAA-3' (SEQ ID 3) and 5'-CAGCTTCCTTTCGGGCTTTGT-3' (SEQ ID 4). DNA was sequenced as a custom service (Agowa, Germany). The vector NTi Suite 10 (Invitrogen, USA) was used to analyze and process the DNA sequence. Protein sequences were aligned using the Clustal W program (Swiss EMBnet node server). The nucleotide sequence of the isolated gene has been deposited in the GenBank database under accession number HQ147786 (Thc_cut2).

Newly transformed Escherichia coli BL21-Gold (DE3) cells were used to inoculate 20 mL LB-medium supplemented with 40 μg/mL kanamycin, and cultured overnight at 37° C. and 160 rpm. Overnight cultures were used to inoculate 200 mL of LB-medium with 40 μg/mL kanamycin to OD600 = 0.1 and incubated until OD600 = 0.6-0.8 was reached. Afterwards, the culture was cooled to 20°C and induced with IPTG at a final concentration of 0.05 mM. Induction was performed at 20°C and 160 rpm for 20 hours. Cells were harvested by centrifugation (20 minutes, 4°C, 3200g).

Cell pellets from 200 mL of cell culture were resuspended in 30 mL of binding buffer (20 mM NaH ₂ PO ₄ *2H ₂ O, 500 mM NaCl, 10 mM imidazole, pH 7.4). The resuspended cells were sonicated 3 times with 3 30-s pulses (Vibra Cell, Sonics Materials, Meryin/Satigny, Switzerland) under ice cooling. The lysate was centrifuged (30 min, 4°C, 4000 g) and filtered through a 0.2 μm membrane. Using a HisTrap FF column The purification system (elution buffer 20mM NaH ₂ PO ₄ *2H ₂ O, 500mM NaCl, 500mM imidazole, pH 7.4) purified the cell lysate. For the characterization of cutinase, the HisTag elution buffer was exchanged with 100 mM Tris HCl pH 7.0 by using a PD-10 desalting column (GE Healthcare).

Protein concentration was determined by Bio-Rad protein detection kit (Bio-Rad Laboratories GmbH) and bovine serum albumin as protein standard. SDS-PAGE was performed corresponding to Laemmli (Laemmli, UK, Nature 1970, 227(5259), 680-685) and proteins were stained with Coomassie brilliant blue R-250.

All chemicals were of analytical grade from Sigma (Germany).

Hydrolysis reaction

The hydrolysis of the plastic article was carried out as follows. In each sample, shake at 300 rpm at 50°C in 1 mL buffer K ₂ HPO ₄ /KH ₂ PO ₄ 100 mM at pH 7.0 in a Thermomixer Comfort (Eppendorf) , 10 mg of plastic products were incubated with 5 μM cutinase for 6 hours to 72 hours. All experiments were similarly performed three times.

i) plastic in buffer without enzyme; ii) enzyme in buffer without plastic were used as controls.

Terephthalic acid (TA) determination method

Following enzyme treatment, proteins were precipitated on ice using 1:1 (v/v) absolute methanol (Merck). The samples were centrifuged (Hettich MIKRO 200R, Tuttlingen, Germany) at 16000 g for 15 minutes at 0°C. The supernatant for the assay was taken into an HPLC vial and acidified by adding 3.5 μL of 6N HCl. The HPLC used was a DIONEX P-580PUMP (Dionex Cooperation, Sunnyvale, USA) with an ASI-100 automatic sample injector and a PDA-100 photodiode array detector. For the analysis of TA, a reversed-phase column RP-C18 (Discovery HS-C18, 5 μm, 150 x 4.6 mm with pre-column, Supelco, Bellefonte, USA) was used. Utilize 60% water, ₁₀ % 0.01N _H2SO4 and 30% methanol as eluent, gradually (15 minutes) change to 50% methanol and 10% acid, gradually (20 minutes) change to 90% methanol and acid, keep 2 minutes, then gradually change to the initial position, run for 5 minutes, and analyze. The flow rate was set at 1 mL/min and the column was maintained at a temperature of 25°C. The injection volume was 10 μL. Detection of TA was performed with a photodiode array detector at a wavelength of 241 nm. Quantitation can be performed using standards of terephthalic acid (Merck code: 800762) diluted 1:1 in buffer:MetOH at different concentrations (1, 5, 10, 50, 100, 250 μM) that The samples were prepared in the same way as the samples.

result

This experiment shows that plastic articles formulated with polymers and additives can be recycled using the method of the invention. Furthermore, in order to increase monomer recovery, it may be advantageous to carry out a mechanical pretreatment of the plastic, which increases the surface contact of the plastic and the enzyme.

More specifically, the PET film was hydrolyzed by cutinase to obtain TA during 72 hours. The longer the reaction time, the more TA was produced (Fig. 2).

Hydrolysis of PTT by cutinase: 4.087 ± 0.122 μM TA was obtained within 24 hours.

Hydrolysis of PET bottles in powder form with a particle size of 1 mm was performed by cutinase: 7.301 ± 0.162 μM TA was obtained within 24 hours. Therefore, the method of the present invention can also be applied to PET plastics formulated with additives, such as those found in plastic waste.

Different sized particles of PET bottle powder were hydrolyzed by cutinase to obtain TA within 24 hours. Reducing the particle size by mechanical milling increased the enzyme efficiency, resulting in more TA: 15.296 ± 1.012 μM TA at a particle size of 250 μm and 7.301 ± 0.162 μM TA at a particle size of 1 mm (Fig. 3 ).

C] Polyamide recycling using enzymes

Thanks to the method according to the invention, plastic articles comprising polyamides can be recycled. This example shows the recovery of adipic acid by treating plastic articles composed of PA with polyamidase expressed by a recombinant strain of E. coli.

Plastics and pretreatment

Commercial polyamide fabric was purchased from Rhodia (Switzerland): PA6,6, 63 g/m2, cut into pieces of 3 cm x 3 cm. It was washed with Na ₂ HPO ₄ , 2H ₂ O, 5 mM for 30 minutes to remove the surface polish.

Polyamidase Manufacturing

The gene encoding the polyamidase from Nocardia IFM 10152 (NCBI deposit number NC 006361) is the following codons optimized for expression in Escherichia coli (GeneArt AG, Germany) and fused to allow expression in the protein. The nucleotide sequence of 6xHisTag was introduced into the C-terminus. According to the manufacturer's instructions, the gene was digested with restriction endonucleases NdeI and HindIII (New England Biolabs, USA), dephosphorylated with alkaline phosphatase (Roche, Germany), purified, and T4 DNA-ligated Enzyme (Fermentas, Germany) was ligated to pET26b(+) (Novagen, Merck KGaA, Germany) and transformed in E. coli BL21-Gold (DE3). The plasmid mini kit from Qiagen (Germany) was used to prepare plasmid DNA. Plasmids and DNA fragments were purified by Qiagen DNA purification kit (Qiagen, Germany). Use the newly transformed cells to inoculate 20 mL of LB-medium supplemented with 40 μg/mL kanamycin. The culture was grown overnight on a rotary shaker at 30°C and 160 rpm. Then, 1 mL of the overnight culture was transferred to a 500 mL shake flask containing 200 mL of the same liquid medium and incubated at 30° C. until an optical density (600 nm) between 0.6 and 0.8 was reached. The cultures were cooled to 20°C and induced with IPTG at a final concentration of 0.05 mM. Induced cultures were incubated overnight at 20°C and 160 rpm. Cells were harvested by centrifugation (3200g, 4°C, 20 minutes).

Purification was performed by gravity flow chromatography using Ni-NTA sepharose according to the manufacturer's protocol (IBA GmbH, Germany), except that 100 mM imidazole was used to elute the protein. For the characterization of polyamidases, the HisTag elution buffer was exchanged with 100 mM Tris HCl pH 7.0 by using a PD-10 desalting column (Amersham Biosciences).

Protein concentration was determined by Uptima BC Assay protein quantification kit from Interchim (France) and bovine albumin as protein standard. SDS-PAGE was performed according to Laemmli (Laemmli, UK, Nature 1970, 227(5259), 680-685) and proteins were stained with Coomassie brilliant blue R-250.

Hydrolysis reaction

The polyamide fabrics were incubated with 2 mg/mL polyamidase in 100 mL citrate phosphate buffer (25 mM, pH 5.0). After hydrolysis, the fabrics were washed with sodium carbonate (9.4 mM, pH 9.5), followed by four rinse steps with distilled water to remove adsorbed proteins. All steps were performed at 30°C for 30 minutes. After the final step, the fabric was dried overnight at room temperature.

i) plastic in buffer without enzyme; ii) enzyme in buffer without plastic were used as controls.

Adipic acid assay

After enzyme treatment, the protein was precipitated using Carrez precipitation. Therefore, the pH of the sample must be between 4 and 6. 2% solution C1 (0.252M K ₄ [Fe(CN) ₆ ] ₃ , H ₂ O) was added to the sample, after vortexing and incubation for 1 min, 2% solution C2 (1 mM ZnSO ₄ , 7H ₂ O) was added . After vortexing and incubation for 5 minutes, the samples were centrifuged (30 minutes, 16000 g, 25°C). The supernatant was directly filtered through a 0.45 μm filter into a glass vial for HPLC analysis (Hewlett Packard Series 1100, Refractive Index Detector: Agilent Series 1100). A column ION-300 (Transgenomic, Inc.) was used, the flow rate was set at 0.1 mL/min and 0.01 N H ₂ SO ₄ was used as the mobile phase. The temperature was set at 45°C, and the injection volume was 40 μL. Detection is achieved at a wavelength of 220 nm. Calibration was achieved using adipic acid standard solution.

result

Hydrolysis of PA by polyamidase: 9.4 μM adipate was obtained within 48 hours.

D] Recycling of Aliphatic Polyesters Using Recombinant Microorganisms

Thanks to the method of the invention, it is possible, as in Example A, to recycle plastic articles comprising aliphatic polyesters, such as PLA, using enzymes and using recombinant microorganisms expressing and secreting depolymerases, which have the ability to prevent the monomers obtained. Improved metabolism of consumption. Improved metabolism can be achieved by gene deletion or by gene disruption or knockout. This experiment shows the recovery of lactic acid by the treatment of plastic articles composed of semi-crystalline PLA with recombinant strains of Lactococcus lactis or E. coli. The strain improvement shown in the Examples can also be performed on other microorganisms.

Plastics and pretreatment

PLA pellets were purchased from NaturePlast (PLLA 001) and ground to a powder with a particle size of less than 500 μm using a universal mill Condux CUM 100.

In an aluminum pan, at a scan rate of 10°C/min, under a nitrogen atmosphere (50mL/min), from -50°C to 300°C, using a Q 100 TA-RCS 90 instrument, for a sample of about 8 mg, using the differential Scanning calorimetry (DSC) tests to determine the glass transition temperature (Tg) and crystallinity of polymers in plastic articles.

PLA powder has a Tg of 59°C and is semi-crystalline with a crystallinity of 14.9%. As determined by SEC, its mass properties are Mw 71000g/mol and Mn 45000g/mol.

Lactococcus lactis construct

According to the classical method described in "Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York", the wild-type strain of L. The gene pld (SEQ ID 5) of PLA depolymerase was recombined (Nakamura et al., 2001-Appl.Environ.Microbiol.67:345-353). Thus, homologous recombination was achieved in the pNZ8048 plasmid (Kuipers et al., 1998-J. Biotechnol. 64:15-21). The recombinant plasmid having the pld gene is called "pNZ-pld". According to the classic method described in "Ho et al., 1995-transformation of Lactococcus by electroporation (Transformation of Lactococcus by electroporation)-Methods Mol. Biol. 47:195-199", the pNZ-pld plasmid pair was electroporated via pNZ-pld plasmid Lactococcus lactis strains were transformed. The recombinant L. lactis strain was called MG1363-pNZ-pld. The negative control corresponds to the Lactococcus lactis MG1363 strain transformed with the empty plasmid pNZ8048.

E. coli construct

Escherichia coli K12-MG1655 contains three kinds of lactate dehydrogenase (LDH). One LDH is specific for the D-lactate isomer. Another LDH converts pyruvate to lactate under anaerobic conditions. The last LDH is specific for the L-lactate isomer and allows growth on this substrate (Haugaard, N. (1959) D- and L-lactate oxidase of Escherichia coli. Biochim Biophys Acta 31, 66-77; Kline, E.5. and Mahler, E.R. (1965). Lactate dehydrogenase from Escherichia coli, Ann N Y Acad Sci 119, 905-917). For the recycling method, the expression of this last LDH must be suppressed in order to recover it without any consumption of lactate.

Disruption of the lldD gene (SEQ ID 6) encoding LDH in E. coli allowed inhibition of lactate consumption. To disrupt the lldD gene, ampicillin ( Amp) The amp resistance gene is inserted in the sequence of the lldD gene, and the primers DlldD-F and DlldD-R have a sequence homologous to the sequence of the lldD gene and a sequence homologous to the amp gene. Amp-resistant transformants were then selected, and the chromosomal structure at the mutation site was verified by PCR analysis and by DNA sequencing using appropriate primers that were compatible with the upstream and downstream sequences of the lldD gene (SEQ ID 9 and SEQ ID 10) homologous.

Then, according to the classical method described in "Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York", E. The gene pld of the PLA depolymerase was recombined (Nakamura et al., 2001 - Appl. Environ. Microbiol. 67:345-353). Thus, homologous recombination was achieved in the pNZ8048 plasmid (Kuipers et al., 1998-J. Biotechnol. 64:15-21). The recombinant plasmid having the pld gene is called "pNZ-pld". The disrupted lldD E. coli strain was transformed with the pNZ-pld plasmid. The recombinant disrupted E. coli strain was designated K12DlldD-pNZ-pld. The negative control corresponds to the E. coli strain transformed with the empty plasmid pNZ8048.

Hydrolysis reaction

At 30°C, in R2 medium, in a 2L bioreactor, two strains of Lactococcus lactis MG1363-pNZ-pld and MG1363-pNZ and two strains of Escherichia coli K12DlldD-pNZ-pld and K12DlldD- pNZ8048 was cultured. Each culture was subdivided into 2 subcultures: batch 1 without PLLA and batch 2 with 0.1% (m/v) PLLA.

Lactic acid assay

After culturing for 2 days, 2 mL of each reaction medium was sampled. Samples were centrifuged at 16000g for 3 minutes at 0°C. The supernatant for analysis was subjected to 0.45 μm filtration and 20 μL was injected into the HPLC. The HPLC used was Ultimate-3000 (Dionex, ThermoScientific) with an autosampler thermostat controlled to 10°C and a column chamber thermostat controlled to 50°C. For the analysis of LA, an Aminex H+HPX-87H column was used. Analysis was performed using ₅ mM _H2SO4 as eluent. The flow rate was set at 0.5 mL/min and the column was maintained at a temperature of 50°C. Detection of LA was performed at 220 nm using a variable wavelength detector. Quantitation was possible considering standards prepared with L-lactic acid (L-1750) from Sigma dissolved in Tris HCl 20 mM pH 8, in the concentration range 0-300 mM.

result

The recombinant Lactococcus lactis MG1363-pNZ-pld expressing only PLA depolymerase and the recombinant disrupted E. coli strain K12DlldD-pNZ-pld produced lactic acid from PLA. Lactate dehydrogenase disruption allows recovery of lactate where it is not consumed by the strain.

Claims (15)

CLAIMS 1. A method for recycling at least one plastic article, said method comprising using an enzyme to depolymerize at least one polymer of said plastic article into monomers and recovering the resulting monomers. 2. The method of claim 1, wherein said enzyme is a degradative enzyme suitable for depolymerizing at least one polymer of said plastic article into monomers. 3. The method of claim 2, wherein said degrading enzyme is cutinase (EC 3.1.1.74), lipase (EC 3.1.1.3), esterase, carboxylesterase (EC 3.1.1.1), p-nitrobenzene Methylesterase, serine protease (EC 3.4.21.64), protease, amidase, aryl acyl amidase (EC 3.5.1.13), oligomer hydrolase such as 6-aminocaproate cyclic dimer hydrolysis Enzyme (EC 3.5.2.12), 6-aminocaproate dimer hydrolase (EC 3.5.1.46), 6-aminocaproate-oligomer hydrolase (EC 3.5.1.B17-.-) , peroxidase, laccase (EC 1.10.3.2). 4. The method according to any one of the preceding claims, comprising contacting said plastic article with at least one microorganism expressing and secreting said enzyme. 5. The method of claim 4, wherein the microorganism is a recombinant microorganism having an improved metabolism that prevents consumption of the resulting monomer. 6. The method according to claim 4 or 5, wherein the microorganism is a recombinant microorganism expressing and secreting a recombinant degrading enzyme. 7. The method of any one of the preceding claims, wherein the plastic article is pretreated prior to degradation. 8. The method of claim 7, wherein said pretreatment comprises mechanical/physical modification of said plastic article, preferably grinding of said plastic article. 9. The method according to any one of the preceding claims, wherein at least one lipophilic and/or hydrophilic agent is used together with the enzyme. 10. The method of any one of the preceding claims, further comprising purifying the monomer and reprocessing the purified monomer. 11. The method according to any one of the preceding claims, wherein the plastic article comprises at least one polymer selected from the group consisting of amorphous and/or semi-crystalline polyesters and polyamides. 12. The method of claim 11, wherein said polyester is selected from the group consisting of: polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate ( PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), poly(L-lactic acid) (PLLA), poly(D-lactic acid) (PDLA), poly(D,L - Lactic acid) (PDLLA), PLA stereocomplex (scPLA), polyhydroxyalkanoate (PHA), poly(3-hydroxybutyrate) (P(3HB)/PHB), poly(3-hydroxyvaleric acid ester) (P(3HV)/PHV), poly(3-hydroxyhexanoate) (P(3HHx)), poly(3-hydroxycaprylate) (P(3HO)), poly(3-hydroxydecanoate ester) (P(3HD)), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)/PHBV), poly(3-hydroxybutyrate-co- -3-hydroxyhexanoate) (P(3HB-co-3HHx)/(PHBHHx)), poly(3-hydroxybutyrate-co-5-hydroxyvalerate) (PHB5HV), poly(3-hydroxy butyrate-co-3-hydroxypropionate) (PHB3HP), polyhydroxybutyrate-co-hydroxyoctanoate (PHBO), polyhydroxybutyrate-co-hydroxyoctadecanoate (PHBOd), Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) (P(3HB-co-3HV-co-4HB)), polybutylene succinate (PBS), Polybutylene Succinate Adipate (PBSA), Polybutylene Adipate Terephthalate (PBAT), Polyethylene Furoate (PEF), Polycaprolactone (PCL), poly(ethylene adipate) (PEA), and blends/mixtures of these materials. 13. The method of claim 11, wherein said polyamide is selected from the group consisting of: polyamide-6 or poly(β-caprolactam) or polycaproamide (PA6), polyamide-6,6 or poly(hexamethylene adipamide ) (PA6,6), poly(11-aminoundecylamide) (PA11), polylauryllactam (PA12), poly(butylene adipamide) (PA4,6), poly(decyl pentamethylenediamide) (PA5,10), poly(hexamethylene azelamide) (PA6,9), poly(hexamethylene sebacamide) (PA6,10), poly(hexamethylene dodecylamide ) (PA6,12), poly(m-xylylene adipamide) (PAMXD6), polyhexamethylene adipamide/polyhexamethylene terephthalamide copolymer (PA66/6T), polyhexamethylene adipamide Hexamethylenediamide/polyhexamethyleneisophthalamide copolymer (PA66/6I), and blends/mixtures of these materials. 14. The method according to any one of the preceding claims, wherein at least two different polymers of the plastic article are depolymerized simultaneously or sequentially. 15. The method according to any one of the preceding claims, wherein at least two plastic articles are recycled simultaneously or sequentially.